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Research Report

Distribution of urocortin 3 neurons innervating the ventralpremammillary nucleus in the rat brain

Judney Cley Cavalcantea, Luciane Valéria Sitaa, Marcelo Betti Mascaroa,b,Jackson Cioni Bittencourta,c, Carol Fuzeti Eliasa,c,⁎aLaboratory of Chemical Neuroanatomy, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, BrazilbNove de Julho University Center-Uninove, São Paulo, BrazilcCenter for Neuroscience and Behavior, Institute of Psychology, University of São Paulo, São Paulo, Brazil

A R T I C L E I N F O

⁎ Corresponding author. Laboratory of ChemiSão Paulo, Av. Prof. Lineu Prestes, 2415, room

E-mail address: cfelias@usp.br (C.F. Elias)

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.03.043

A B S T R A C T

Article history:Accepted 15 March 2006

Urocortin 3 (Ucn 3) is a recently described peptide of the corticotropin-releasing factorfamily. Neurons expressing Ucn 3 mRNA and peptide are distributed in specific brain areas,including themedian preoptic nucleus, the perifornical area (PFx), and themedial nucleus ofthe amygdala (MEA). Fibers immunoreactive to Ucn 3 are confined to certain brain nuclei,being particularly dense in the ventral premammillary nucleus (PMV). In studies involvingelectrolytic lesions and analysis of Fos distribution according to behavioral paradigms, thePMV has been potentially implicated in conspecific aggression and sexual behavior.However, the role that Ucn 3 plays in this pathway has not been explored. Therefore, weinvestigated the origins of the urocortinergic innervation of the PMV of Wistar rat in anattempt to map the brain circuitry and identify likely related functions. We injected theretrograde tracer cholera toxin b subunit into the PMV and found that 88% of the Ucn 3-immunoreactive fibers in the PMV originate in the dorsal MEA, and that few originate in thePFx. As a control, we injected the anterograde tracer biotin dextran amine into both regions.We observed that the PMV is densely innervated by the MEA, and scarcely innervated by thePFx. The MEA is a secondary relay of the vomeronasal system and projects amply tohypothalamic nuclei related to hormonal and behavioral adjustments, including the PMV.Although physiological studies should also be performed, we hypothesize that Ucn 3participates in such pathways, conveying sensory information to the PMV, which in turnmodulates behavioral and neuroendocrine responses.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Medial nucleus of amygdalaPerifornical areaOdorPheromone

1. Introduction

Urocortin 3 (Ucn 3), also known as stresscopin (Hsu and Hsueh,2001), isa recentlydescribedpeptideof thecorticotropin-releasingfactor (CRF) family and is a selective ligand for CRF type 2 (CRF2)receptors (Lewis et al., 2001). It has been reported that Ucn 3

cal Neuroanatomy, Depar105, São Paulo 05508-90

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er B.V. All rights reserved

modulates stress response, as well as feeding and autonomicactivity (Bale et al., 2002; Hashimoto et al., 2004; Lewis et al., 2001;Valdez et al., 2003). However, exactly which brain pathways areinvolved in these functions is a matter of speculation.

Distribution of Ucn 3 mRNA and immunoreactive cellbodies in the rat brain is limited, restricted mainly to the

tment of Anatomy, Institute of Biomedical Sciences, University of0 SP, Brazil.

.

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median preoptic nucleus, the anterior perifornical area, theprincipal division of the bed nucleus of the stria terminalis andthemedial nucleus of the amygdala (MEA) (Lewis et al., 2001; Liet al., 2002). Distribution of Ucn 3-immunoreactive (Ucn 3-ir)fibers is particularly dense in the lateral septal nucleus, theventromedial nucleus of the hypothalamus, and the ventralpremammillary nucleus (PMV) (Li et al., 2002). The PMV islocated in the posterior levels of the medial hypothalamus, aregion that has been designated the “behavior controlcolumn” (Swanson, 2000). The results of studies involvingelectrolytic lesions of the premammillary area have suggestedthat the PMV participates in the regulation of aggressivebehavior (van den Berg et al., 1983). In addition, studies usingFos protein as a marker for neuronal activity have shown thatthe PMV is activated following copulation or conspecificaggression paradigm (Kollack-Walker and Newman, 1995;Veening et al., 2005). However, it has been shown thatolfactory stimulation is sufficient to induce Fos in the PMVof males exposed to soiled bedding cages where cycling

Fig. 1 – Distribution of urocortin 3-immunoreactive (Ucn 3-ir) fibefield photomicrographs showing two rostrocaudal levels of the Pphotomicrographs showing the distribution of Ucn 3-ir in two rophotomicrographs showing Ucn 3-ir varicosities (double arrows)panel F, terminal-like structures are in close apposition with PMnucleus; fx, fornix; PMD, dorsal premammillary nucleus. Scale b

females have been housed (Yokosuka et al., 1999), suggestingthat the PMV plays an important role in pheromonediscrimination.

Canteras et al. (1992) showed that the PMV possessesbidirectional connections with sexually dimorphic nuclei, aswell as with areas such as the anteroventral periventricularnucleus, the medial preoptic nucleus, the bed nucleus ofstria terminalis, and the MEA, all of which are related toreproductive control and luteinizing hormone (LH) secre-tion. In fact, we have recently shown that fibers originatingin the PMV form close appositions with gonadotropin-releasing hormone (GnRH) in male and female rats (Rondiniet al., 2004). Therefore, the PMV is apt to modulate LHsecretion.

The PMV neurons also express the long form of leptinreceptor (ObRb) mRNA and respond to circulating leptin (Eliaset al., 2000, 2001; Elmquist et al., 1998), a hormone secreted byadipocytes. Leptin has profound effects on feeding, as well ason metabolic and neuroendocrine homeostasis (Zhang et al.,

rs in the ventral premammillary nucleus (PMV). (A, C) Bright-MV in reference (thionin staining) sections. (B, D) Dark-fieldstrocaudal levels of the PMV. (E, F) Bright-fieldand terminal-like structures (single arrows). Note that, inV neurons. Abbreviations: 3v, third ventricle; Arc, arcuatear: A–D = 400 μm; E–F = 50 μm.

Fig. 2 – Line drawings showing the injection site of the retrograde tracer cholera toxin b subunit (CTb) centered in the ventralpremammillary nucleus (PMV). (A, B) Schematic drawings of four cases in which the CTb injections were considered good.Abbreviations: 3v, third ventricle; Arc, arcuate nucleus; fx, fornix.

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1994). Individuals deficient in leptin production (ob/ob) orleptin response (db/db) are obese and infertile, suggestingthat leptin is a critical indicator linking adiposity andreproduction (Ahima et al., 2000; Casanueva and Dieguez,1999). It is well accepted that most leptin functions arecontrolled by receptors located in the brain (Cohen et al.,2001). Among the hypothalamic nuclei that express the ObRb,the PMV is the only one that, to date, has not been related toenergy balance and body weight regulation (Elmquist et al.,1999). The PMV also expresses high levels of gonadal steroidreceptors (Simerly et al., 1990), suggesting that it is welllocated to integrate nutritional and reproductive information.These findings motivated us to investigate the origins of thedense Ucn 3 innervation of the PMV. We attempted tocharacterize the underlying circuitry in order to contributeto the understanding of the role that stress-related peptides

Fig. 3 – Urocortin 3 (Ucn 3) neurons located in the medial nucleusnucleus (PMV). (A) Bright-field photomicrograph showing the injecentered in the PMV (case W286). (B) Bright-field photomicrograp(retrograde-labeled neurons) in the posterodorsal part of the MEA3-immunoreactive (Ucn 3-ir) neurons that are also CTb immunorventricle; Arc, arcuate nucleus; fx, fornix; opt, optic tract. Scale b

play in brain pathways involved in the control of behavioralresponses.

2. Results

2.1. Distribution of urocortin 3 immunoreactivity

We observed Ucn 3-ir cell bodies in several areas in the brain,in agreement with previous descriptions of the use ofimmunohistochemical and in situ hybridization methods(Lewis et al., 2001; Li et al., 2002). Few Ucn 3-ir cell bodieswere found in the median preoptic nucleus. Intensely stainedcells were observed in the PFx between the fornix and theparaventricular nucleus of the hypothalamus (PVH), and veryfew cells were found within the borders of the PVH. Outside

of the amygdala (MEA) innervate the ventral premammillaryction site of the retrograde tracer cholera toxin b subunit (CTb)h showing the distribution of CTb-immunoreactive neurons(MEApd). (C, D) Fluorescent photomicrographs showing Ucneactive (CTb-ir) in the MEApd. Abbreviations: 3v, thirdar: A = 400 μm; B = 200 μm; C and D = 100 μm.

Fig. 4 – Line drawings showing the distribution of dual-labeled neurons in the anterior perifornical area (PFx) and in themedialnucleus of the amygdala (MEA). (A–C) Schematic drawings of two rostrocaudal levels of the PFx (A), of two rostrocaudal levels ofthe anterodorsal MEA (MEAad, B) and of two rostrocaudal levels of the posterodorsal MEA (MEApd, C), showing the distributionof urocortin 3-immunoreactive (Ucn 3-ir) neurons (filled circles), cholera toxin b subunit-immunoreactive (CTb-ir) neurons(open circles), and dual-labeled neurons (x). Abbreviations: 3v, third ventricle; BAOT, bed nucleus of the accessory olfactorytract; fx, fornix; ic, internal capsule, LHA, lateral hypothalamic area; MEAav, anteroventral part of the medial nucleus of theamygdala; MEApv, posteroventral part of the medial nucleus of the amygdala; opt, optic tract; ox, optic chiasm; PVH,paraventricular nucleus of the hypothalamus; SCh, suprachiasmatic nucleus.

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the hypothalamus, Ucn 3-ir neurons were foundmainly in thedorsal division of the MEA and in the principal division of thebed nucleus of the stria terminalis (BSTpr). In the anterodorsalpart of theMEA (MEAad), Ucn 3-ir neuronswere found near theoptic tract. In the posterodorsal part of the MEA (MEApd), Ucn3-ir neurons were scattered throughout the nucleus, reachingthe most lateral aspect near the stria terminalis.

Immunohistochemical staining showed axons and vari-cosities, in detail, in several subcortical areas. The distributionof Ucn 3 fibers found in the present study is also in agreementwith that reported in prior studies (Li et al., 2002). In thehypothalamus, the densest distribution of Ucn 3-ir fibers andterminals was seen in the ventromedial nucleus of thehypothalamus and in the PMV. In the PMV, we found Ucn 3-ir fibers throughout the nucleus, and the concentration wasapparently higher in the dorsal aspect near the fornix (Figs.1A–D). Overall, the fibers present numerous varicosities and

terminal-like aspects, with buttons widely distributed withinthe nucleus (Figs. 1E–F).

2.2. MEA and PFx urocortin 3 neurons project to the PMV

In order to determine the origin of Ucn 3 fibers, we injectedthe retrograde tracer CTb into the PMV. We obtained 4 casesthat were considered good (injections centered in the PMV).In two cases (W282 and W286), the injections were limited tothe PMV. In the other two cases (W276 and W 281), theinjections were centered in the PMV but extended ventrally,slightly contaminating the lateral portion of the arcuatenucleus (Figs. 2A–B and 3A). In every case, we foundretrograde-labeled neurons in the medial preoptic nucleus,the arcuate nucleus, the ventromedial nucleus of thehypothalamus, and the ventral division of the MEA, as hasbeen previously described (Canteras et al., 1992). In areas

Table 1 – Quantification of retrogradely (cholera toxin bsubunit) labeled neurons expressing urocortin 3 in tworostrocaudal levels of each region (1 and 2)

Regions Atlaslevel

TotalUcn 3-ir

Doubles CTb+ Ucn 3

% Doubles/total Ucn 3

PFx1 23 21.6 ± 3.5 1.3 ± 0.3 3.8 ± 0.3PFx2 25 19.3 ± 4.2 0.3 ± 0.3 1.5 ± 1.5MEAad1 26 10.3 ± 1.8 1.3 ± 0.3 12.6 ± 0.9MEAad2 27 10 ± 2.1 2.3 ± 0.6 22.3 ± 3.1MEApd1 29 17 ± 1.5 4.3 ± 0.8 25.1 ± 3.2MEApd2 30 12.6 ± 1.2 3.3 ± 0.3 27.1 ± 4.8

Values represent estimates of the mean cell counts ± SEM (n = 3).The atlas level designations correspond to those described byPaxinos and Watson (1998).Abbreviations: Ucn 3, urocortin 3; CTb, cholera toxin b subunit; PFx,perifornical area; MEAad, anterodorsal part of the medial nucleusof the amygdala; MEApd, posterodorsal part of the medial nucleusof the amygdala.

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expressing Ucn 3 immunoreactivity, we found moderatenumbers of retrograde-labeled neurons in the BSTpr, MEAad,and MEApd (Fig. 3B), whereas few retrograde-labeled neu-rons were observed in the PFx.

In the BSTpr, very few dual-labeled neurons were found. Infact, Ucn 3-ir neurons were located lateral to the retrograde-labeled neurons. In the PFx, we observed few retrograde-labeled neurons expressing Ucn 3 immunoreactivity. Thequantification of two rostrocaudal levels revealed that ap-proximately 3% of the Ucn 3-ir neuronswere dual labeled (Figs.4A–B, Table 1). In the MEA, we observed higher numbers ofdual-labeled neurons (Figs. 4C–F). Following the quantificationof two rostrocaudal levels of the MEAad and of the MEApd, wefound that approximately 17% and 26%, respectively, of theUcn 3-ir neurons were also immunoreactive to the retrogradetracer CTb (Figs. 3C–D, Table 1). Of the total number of dual-labeled neurons, around 12% were in the PFx, 28% were in theMEAad, and 60% were in the MEApd.

2.3. PFx and MEA neurons innervate the PMV

As a control, we injected the anterograde tracer BDA into thePFx and the MEA, subsequently investigating the occurrenceof varicosities and apparent terminals within the PMV. Weobtained 6 cases with injections favorable for evaluation (2centered in the PFx and 4 centered in the dorsal MEA). One ofthe injections into the PFx was located between the fornix andthe PVH, slightly contaminating the anterior hypothalamicnucleus and the PVH. The other injection into the PFx wasposterior with slight contamination of the anterior hypotha-lamic nucleus (Figs. 5A and 6). The analysis of these casesshowed that PFx projections to the PMV are sparse, with alimited concentration predominantly in the dorsal portion ofthe nucleus (Fig. 5B). In general, such fibers presentedvaricosities and terminal-like aspect (Fig. 5C).

In the MEA, we obtained 4 cases in which the tracer wasrestricted to the limits of the nucleus and that were thereforeconsidered good (Figs. 5D and 7). In two cases (W659 andW772), the injections were located near the optic tract in themedial aspects of the MEApd, slightly contaminating theMEAad. In the third case (W660), the BDA was located in the

lateral portion of the MEApd. In the fourth case (W773), theBDA injection was more widespread, encompassing the fullextent of the dorsal MEA. In agreement with previous reports(Canteras et al., 1995; Coolen and Wood, 1998), we found BDAvaricose fibers in the medial preoptic nucleus, in the BSTpr, inthe lateral aspects of the arcuate nucleus, in the ventromedialnucleus of the hypothalamus, and in the PMV. The concen-tration and distribution of the projections were consistentamong cases, although we observed apparent differentialinnervation of the PMV. In the cases in which the BDAinjection site was immediately adjacent to the optic tract, wefound fibers dispersed throughout the nucleus. In the case inwhich the injection site was more lateral, fibers wereconcentrated in the dorsal aspect of the PMV (Fig. 5E). Inaddition, we observed dense distribution of varicosities andterminal-like structures in every case analyzed. These vari-cosities were in close apposition with PMV neurons (Fig. 5F).

3. Discussion

We observed that Ucn 3-ir fibers are densely distributedthroughout the PMV, confirming the findings of previousstudies. In the present study, we demonstrate that thisinnervation originates predominantly in the dorsal part ofthe MEA, and that little innervation originates in theperifornical area.

Because the Ucn 3 is a member of the CRF family ofpeptides and is a restricted CRF2 ligand, most studies havefocused on stress and anxiety responses (Bale and Vale, 2004).Although the CRF type 1 (CRF1) receptor has beenmore clearlyassociated with stress activation and anxiety-like behavior(Takahashi, 2001), there is evidence that CRF2 is also involved.Administration of CRF2 antagonist has been shown to induceanxiety-like behavior and to increase stress sensitivity (Pel-leymounter et al., 2002; Radulovic et al., 1999; Takahashi et al.,2001). The same effects were observed in studies involvingCRF2-deficient mice (Bale et al., 2000; Bale and Vale, 2004;Coste et al., 2000; Kishimoto et al., 2000). Although it is notknownwhich brain pathways are involved in these responses,some authors have suggested that CRF2 dampens or regulatesthe stress response associated with CRF activation (Venihakiet al., 2004). Such regulation might occur through theengagement of specific brain pathways and neuromodulators.The role that Ucn 3 plays in these responses is still a matter ofdebate. Studies in which the elevated plus-maze test was usedhave yielded contradictory results. Various research groupshave shown that Ucn 3 inhibits anxiety-like behavior, whereasothers have shown no such inhibition (Pelleymounter et al.,2004; Valdez et al., 2003; Venihaki et al., 2004). Nevertheless,other authors have shown that Ucn 3 exhibits anxiolytic andlocomotor suppressive effects (Ohata and Shibasaki, 2004;Valdez et al., 2003; Venihaki et al., 2004). These effects mightbe mediated by CRF2 receptors, although the brain circuitryinvolved remains unclear. The PMV is an interesting candidatebecause it displays dense Ucn 3 innervation. However, CRF2 isnot expressed in PMV neurons (Van Pett et al., 2000),suggesting that there is an additional CRF receptor subtype,and that there is another, as yet uninvestigated, functioninvolving this pathway.

Fig. 5 – Neurons in the anterior perifornical area (PFx) and posterodorsal part of the medial nucleus of the amygdala (MEApd)innervate the ventral premammillary nucleus (PMV). (A, D) Bright-field photomicrograph showing the injection site of theanterograde tracer biotin dextran amine (BDA) in the PFx (A) and MEApd (D). (B, E) Dark-field photomicrographs showing thedistribution of BDA fibers innervating the PMV that originate in PFx (B) and MEApd (E) neurons. (C, F) Bright-fieldphotomicrographs showing BDA varicosities and terminal-like structures (arrows) in the PMV from neurons located in the PFx(C) and MEApd (F). Abbreviations: 3v, third ventricle; Arc, arcuate nucleus; fx, fornix; PVH, paraventricular nucleus of thehypothalamus. Scale bar: A, D = 400 μm; B, E = 200 μm; C, F = 50 μm.

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Ucn 3 innervation of the PMV originates in the anterior PFxand dorsal MEA. The PFx is usually considered to be part of thelateral hypothalamic area and surrounds the fornix through-out the tuberal hypothalamus (Paxinos and Watson, 1998).Neurons in the PFx express orexigenic peptides, including themelanin-concentrating hormone (MCH) and orexin/hypocre-tin, and the PFx has been primarily linked to feeding control(Bittencourt et al., 1992; de Lecea et al., 1998; Qu et al., 1996;Sakurai et al., 1998; Sweet et al., 1999). Intracerebroventricularinjections of MCH and orexin/hypocretin increase food intake,and themRNA levels of both are elevated during fasting (Qu etal., 1996; Sakurai et al., 1998). In addition, injections ofneuropeptide Y, calcitonin, and N-methyl D-aspartate agonistin the PFx increase food intake (Chait et al., 1995; Currie andCoscina, 1995; Jolicoeur et al., 1995; Lee and Stanley, 2005;Stanley et al., 1993). Whether the PFx-PMV urocortinergicpathway plays a role in regulating feeding behavior remainsunknown, but it is intriguing to speculate that it might beinvolved in the modulation of nutritional information. The

PMV expresses a great amount of ObRb mRNA (Elmquist et al.,1998) and respond to circulating leptin, a hormone signalingthe amount of energy stored (Flier, 1998; Zhang et al., 1994).Like other members of the CRF family, Ucn 3 is an anorexi-genic peptide (Hashimoto et al., 2004; Hsu and Hsueh, 2001;Ohata and Shibasaki, 2004; Pelleymounter et al., 2004).Therefore, the Ucn 3 innervation of the PMV originating inthe PFx, albeit slight, might participate in the modulation ofsuch information. Further studies will be needed in order totest this hypothesis.

The MEA is a secondary relay of the vomeronasal systemand presents dense expression of gonadal steroid receptors,integrating chemosensory information, and hormonal cues(Rasia-Filho et al., 1991; Scalia andWinans, 1975; Simerly et al.,1990; Wood and Coolen, 1997; Wood and Newman, 1995). Si-milarly, various studies have shown that the MEA expressesFos protein after pheromonal exposition or sexual behavior(Bressler and Baum, 1996; Coolen et al., 1996; Kollack-Walkerand Newman, 1995; Veening and Coolen, 1998) .

Fig. 6 – Line drawings showing the injection site of theanterograde tracer biotin dextran amine (BDA) centered inthe anterior perifornical area (PFx). (A, B) Schematic drawingsof two cases in which the BDA injections were consideredgood (filled circles represent neurons that captured thetracer). Abbreviations: 3v, third ventricle; AHN, anteriorhypothalamic nucleus; Arc, arcuate nucleus; fx, fornix; LHA,lateral hypothalamic area; opt, optic tract; PVH, paraventri-cular nucleus of the hypothalamus; VMH, ventral nucleus ofthe hypothalamus.

Fig. 7 – Line drawings showing the injection site of theanterograde tracer biotin dextran amine (BDA) centered inthe posterodorsal part of the medial nucleus of the amygdala(MEApd). (A–D) Schematic drawings of four cases in whichthe BDA injections were considered good (filled circlesrepresent neurons that captured the tracer). Abbreviations:LHA, lateral hypothalamic area; MEApv, posteroventral partof the medial nucleus of the amygdala; opt, optic tract.

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Electrochemical stimulation of the MEA enhances LH releasein ovariectomized, estrogen-primed female rats and precipi-tates the LH peak on the proestrous day (Beltramino andTaleisnik, 1978). Meredith and Westberry (2004) showed thatthe dorsal MEA is important for the discrimination betweensocially relevant (conspecific) and socially irrelevant (hetero-specific) chemosensory stimuli in male hamsters (Meredithand Westberry, 2004). In addition, female pheromones induceFos expression in the medial aspect of the male MEApd(Meredith and Westberry, 2004), whereas neurons in thelateral MEApd are activated after mating that involvesejaculation (Pfaus and Heeb, 1997). Herein, we found that,albeit apparently more lateral, Ucn 3 neurons are notrestricted to a particular region within the MEApd, makingthe evaluation of a likely role according to its distributioninappropriate. Further experiments will be needed in order toinvestigate the actual participation of Ucn 3 in these functions.Similarly, the PMV also expresses Fos after exposure to femalepheromones (Yokosuka et al., 1999), a response that isprobably triggered by MEA projections because the PMV doesnot receive direct innervation from the main or the accessoryolfactory nuclei (Scalia and Winans, 1975). Lesion of the PMVblocks the increase in LH release in ovariectomized, estrogen-primed female rats and prevents the precipitation of the LHpeak during proestrus induced by MEA or accessory olfactorybulb electrochemical stimulation or by pheromone exposition(Beltramino and Taleisnik, 1985). Although physiologicalstudies are still needed, we hypothesize that Ucn 3 is part ofthis circuitry, conveying olfactory information to the PMV.

The PMV and MEA are both also activated followingagonistic encounters (Kollack-Walker and Newman, 1995;Luiten et al., 1985; van den Berg et al., 1983), and a recentstudy showed that Ucn 3 may participate in the modulation ofmaternal defensive behavior (D'Anna et al., 2005). Certainly,both situations involve pheromone-triggered arousal andincreased stress response. We propose that the MEA–PMVpathway is engaged in these responses, and that Ucn 3 has thepotential tomediate the physiological adaptations observed inaggressive/defensive behavior.

In summary, neurons in the MEA respond to olfactorystimulation and project to the PMV, where information (from

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the PFx and from leptin) regarding nutrition is integrated withinformation regarding reproductive status. Together, theseinputs may induce the adaptations necessary for the appro-priate behavior to occur. Our findings suggest that Ucn 3 is aprime candidate for mediating the pathways underlying thisintegrative function.

4. Experimental procedures

4.1. Animals

Normal adult male Wistar rats (280–320 g) were housed twoper cage in the animal care facility of our institution.Animals were maintained on a 12-h light/dark cycle (lightson at 7 am) in a temperature-controlled environment (21 ± 2 °C)and were given free access to food and water. All experimentswere carried out in accordance with the guidelines estab-lished by the National Institutes of Health Guide for theCare and Use of Laboratory Animals (1996) and by theUniversity of São Paulo Committee for Research andAnimal Care. We attempted to minimize the number ofrats used, and every effort was made to ensure that no ratsuffered unnecessarily.

4.2. Urocortin 3 immunohistochemistry

Four rats were deeply anesthetized with intraperitonealinjection of 35% chloral hydrate (1 ml) and perfused transcar-dially with saline followed by 4% formaldehyde in boratebuffer (pH 9.5 at 4 °C, 900 ml over 25 min). Brains wereremoved, postfixed in the same fixative for 2 h, andcryoprotected overnight at 4 °C in 0.1 M phosphate-bufferedsaline (PBS), pH 7.4, containing 30% sucrose. The brains werecut in the frontal plane into 30-μm sections on a freezingmicrotome. Five series were collected in antifreeze solutionand stored at −20 °C.

One series from each animal was submitted to a standardimmunoperoxidase reaction. Sections were pretreated withhydrogen peroxide and blocked in 2% normal donkey serum(Jackson Laboratories,West Grove, PA, USA) and 0.3% Triton X-100 (Sigma). The sections were then incubated with antiseraagainst Ucn 3 (1:15,000, rabbit anti-human Ucn 3, #6570, kindlyprovided by Drs. Wylie W. Vale and Joan Vaughan) overnightat room temperature. This was followed by incubation for 1 hin biotin-conjugated donkey anti-rabbit IgG (1:1,000, JacksonLaboratories) and for 1 h in avidin–biotin complex (1:500,Vector Labs, Burlingame, CA, USA). The tissue was thensubmitted to immunoperoxidase reaction using 3,3′-diamino-benzidine tetrahydrochloride (DAB; Sigma) as chromogen and0.03% hydrogen peroxide dissolved in 0.02 M potassium PBS(KPBS), pH 7.4, for 2–3 min. The reaction was terminated withrinses in KPBS and intensified using a silver-gold procedure(De Lacalle et al., 1994).

Sections were mounted onto gelatin-coated slides, dehy-drated, delipidated, and coverslipped with DPX mountant(BDH, Poole, England). The antisera specificity and adsorptioncontrol tests had been performed in advance (Li et al., 2002).One adjacent series was submitted to thionin staining andused as reference.

4.3. Retrograde tracer injection

Twenty-four rats were anesthetized with subcutaneous injec-tion of a solution containing ketamine (5 mg/100 g), xylazine(1mg/100 g), and acepromazine (0.2 mg/100 g). Those rats thenreceived stereotaxic injection of the retrograde tracer choleratoxin b subunit (CTb 1%; List Biological Laboratories, Campbell,CA, USA) into the PMV. The CTbwas injected iontophoreticallyfrom a glass micropipette (10–20 μm of the tip internaldiameter) by applying a +5 μA current pulsed at 7-s intervalsover 10–12min. After 15 days, the animals were perfused, afterwhich the brains were dissected, cryoprotected, and sectionedas described.

One series from each animal was submitted to immuno-peroxidase reaction as previously described, using anti-CTbprimary antisera raised in goat (1:50,000; List BiologicalLaboratories, Campbell, CA, USA) and DAB as chromogen.Sections were mounted onto gelatin-coated slides, driedovernight, dehydrated in ethanol, cleared in xylene, andcoverslipped with DPX. Cases considered good (injectioncentered in the PMV) were submitted to dual-label immuno-fluorescence for identification of retrograde-labeled neuronsexpressing Ucn 3 immunoreactivity. Series were incubated inanti-CTb (1:3,000) with 0.3% Triton X-100 and 2% normaldonkey serum, overnight, at room temperature, and influorescein isothiocyanate (FITC)-conjugated IgG donkeyanti-goat (1:200, Jackson Laboratories) for 1 h in KPBS with0.3% Triton X-100 and normal donkey serum. Subsequently,the tissue was submitted to a second immunofluorescenceprocedure, using the Ucn 3 antisera (1:2,000) as the primaryantisera and Cy3-conjugated IgG donkey anti-rabbit (1:200,Jackson Laboratories) as the secondary antisera. Sections weremounted onto gelatin-coated slides and coverslipped withglycerol mountant. Areas containing neurons expressing Ucn3 were analyzed. In two sections per area, Ucn 3-ir neuronsand dual-labeled cell bodies were counted. Two differentanalyses were performed: (1) the percentage of the mean(±SEM) number of dual-labeled cells in relation to the totalnumber of Ucn 3-ir neurons in each area, and (2) thepercentage of the mean (±SEM) number of dual-labeled cellsin each area in relation to the total number of dual-labeledcells throughout the forebrain.

4.4. Anterograde control

Ten adult male rats were anesthetized with subcutaneousinjection of a cocktail containing ketamine, xylazine, andacepromazine as described. The rats then received stereo-taxic injection of biotinylated dextran amine (BDA, 10% in0.01 M PBS; 10,000 MW; Molecular Probes, Eugene, OR, USA)into the anterior perifornical area (PFx) or the dorsal divisionof MEA. The BDA was injected iontophoretically from a glassmicropipette (10–20 μm of the tip internal diameter) byapplying a +5 μA current pulsed at 7-s intervals over 15–20 min. After 10–15 days, the animals were perfused, afterwhich the brains were dissected, cryoprotected, and sec-tioned in the frontal plane. One series from each animal wassubmitted to a 1-h incubation with avidin–biotin complex(Vector), followed by peroxidase reaction using DAB aschromogen. The reaction was intensified using a silver-gold

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procedure (De Lacalle et al., 1994), and the sections werecounterstained with thionin.

4.5. Data analysis and production of photomicrographs

The photomicrographs were captured with a SPOT RT® digitalcamera (Diagnostic Instruments Sterling Heights, MI, USA),adapted to a Leica DMRmicroscope (Leica, Wetzlar, Germany),and a Dell Dimension 4400 computer. Images were digitalizedusing ImagePro Plus®, and Adobe Photoshop 7.0 image-editingsoftware was used to integrate photomicrographs into plates.Only sharpness, contrast, and brightness were adjusted.Drawings were produced using a camera lucida adapted to theLeicamicroscope, and Canvas 6.0 software (Deneba,Miami, FL,USA) was used to assemble the images into plates.

Acknowledgments

We are grateful to Drs. Wylie Vale and Joan Vaughan for theurocortin 3 antisera. We thank Joelcimar Martins da Silva forthe expert technical assistance given and Jefferson Boyles forediting the manuscript. Financial support was provided in theform of grants and fellowships from FAPESP (00/11569-4, 03/06022-4, 04/00585-0, 05/50951-5). JCB and CFE are CNPqinvestigators.

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